Stereocomplexation is one of several approaches for improving polylactide (PLA) properties. The high molecular weight of poly L-lactide (PLLA) and poly D-lactide (PDLA) homopolymers are a constraint during the formation of stereocomplex PLAs (s-PLAs). The presence of s-PLA particles in PLA PLLA/PDLA blends can initiate the formation of s-PLA crystalline structures. We used the solution casting method to study the utilization of s-PLA materials from high molecular weight PLLA/PDLA blends for increasing s-PLA formation. The s-PLA particles initiated the formation of high molecular weight PLLA/PDLA blends, obtaining 49.13% s-PLA and 44.34% of the total crystalline fraction. In addition, the mechanical properties were enhanced through s-PLA crystalline formation and the increasing of total crystallinity of the PLLA/PDLA blends. The s-PLA particles supported initiation for s-PLA formation and acted as a nucleating agent for PLA homopolymers. These unique characteristics of s-PLA particles show potential to overcome the molecular weight limitation for stereocomplexation of PLLA/PDLA blends. Environmental concerns have driven attention toward developing degradable polymers and recycling non-degradable polymers. Replacing fossil fuel-based polymers with degradable polymers and recyclable or reusable non-degradable polymers would help mitigate environmental problems. Polylactide (PLA) is an economically feasible and bio-based polymer that has the potential to replace non-degradable polymers. PLA is biodegradable and biocompatible ,2. Howevw) range of approximately 125,000–150,000 obtained about 25.9% stereocomplexation degree [w, at which only stereocomplex crystallites form, is approximately 1.0 × 105 [The formation of s-PLA was discovered in an enantiomeric polymer blend of poly L-lactide (PLLA) and poly D-lactide (PDLA) with a minimum of seven units of lactide fragment ,4. Previn degree . As Tsujm) of s-PLA is approximately 50 °C higher than the Tm of either PLLA or PDLA [The nucleation of s-PLA crystallites is driven by hydrogen bonding between PLLA and PDLA fragments, resulting in different crystal structures of s-PLA compared to its homopolymers . Differe or PDLA ,16. Prop or PDLA ,16. The  or PDLA ,20 and m or PDLA ,22,23. T or PDLA ,20. In o or PDLA ,22,23.Because of their unique characteristics, s-PLA crystallites have potential as nucleating agents. They are also expected to overcome high-molecular-weight challenges in s-PLA formation from PLLA/PDLA blends. In this work, we were interested in studying the effect of s-PLA nucleation on the stereocomplex formation of PLA from high molecular weight PLLA and PDLA homopolymers through the solution casting method. The presence of s-PLA particles in high molecular weight PLLA/PDLA blends may stimulate the formation of s-PLA and also influence the change in the crystallinity degree of the blends. We evaluated the degree of s-PLA formation from PLLA/PDLA blends, assessed any improvements in mechanical properties, and studied the effect of s-PLA particle size.2)  and 1-dodecanol (DoOH)  were purified by distillation under reduced pressure and dissolved in dry toluene. The toluene  was dried by refluxing it over a benzophenone-Na complex and distilling it in a nitrogen atmosphere immediately prior to use. Chloroform and methanol  were used as received. The s-PLA materials were synthesized from PDLA  and PLLA  blends with a 1:1 weight ratio using supercritical carbon dioxide–dichloromethane at 65 °C and 350 bar for 5 h [The lactide monomers L-lactide (L-LA) and D-lactide (D-LA) were purchased from Corbion . Tin(II)bis(2-ethylhexanoate) and stannous octoate (Sn(Oct)High molecular weight PLLA and PDLA were synthesized through ring-opening bulk polymerization of L-LA and D-LA using stannous octoate  and 1-dodecanol (initiator) at 130 °C for 24 h. All polymerization preparation was carried out in a drying room to control humidity. The ring-opening polymerization was conducted in a round-bottom flask and completed with a magnetic stirrer and oil bath. First, the L-LA or D-LA monomers (20 g), catalyst, and initiator were added to the flask in a ratio of 1500:1:1. To remove any contaminants and moisture, the flask was purged with nitrogen gas three times and vacuumed for 6 h. The flask was then sealed under vacuum conditions and immersed in an oil bath at the desired temperature (130 °C). After polymerization was complete, the polymer products were dissolved in chloroform, precipitated in methanol, filtered, and dried in an oven at 40 °C for 24 h to obtain purified high molecular weight PLLA or PDLA.The stereocomplexation of PLA materials was prepared by combining the PLLA and PDLA homopolymers (1:1 weight ratio) using the solution casting method in the presence of s-PLA particles of various contents and particle sizes. We denoted samples of produced s-PLA as PLLA/PDLA_Ax, PLLA/PDLA_Bx, and PLLA/PDLA_Cx depending on their particle size category . The x values represented the s-PLA content in the blends. The PLLA/PDLA_A1, PLLA/PDLA_A3, PLLA/PDLA_A5, PLLA/PDLA_A10, and PLLA/PDLA_A20 represented the PLA blends with 1%, 3%, 5%, 10%, and 20% s-PLA particle content, respectively. The original PLLA and PDLA blend was used as control material. The PLLA, PDLA, and s-PLA particle mixture was dissolved in dichloromethane with a total polymer to a total solvent ratio (weight to volume) of about 5:100. The mixture was vigorously stirred for 4 h and poured into a Petri glass. It then underwent evaporation at room temperature for 24 h and was subsequently placed in vacuum conditions at 80 °C for 48 h to obtain dry film of the polymer mixture.αK source , a quartz monochromator, and a goniometric plate with an observation range of 3–30° of 2θ.The polymerization conversion of PLA homopolymer was measured via gravimetric analysis. The molecular weight of the purified PLLA and PDLA homopolymers were evaluated using gel permeation chromatography  at 40 °C, with chloroform as a solvent, a flow rate of 1.0 mL/min, and a polymer concentration of 0.2%. The monodisperse polystyrene standard was used for calibration. The synthesized PLLA/PDLA blend films were characterized to evaluate the effect of adding s-PLA materials on the degree of s-PLA formation, as well as on the mechanical and thermal properties. Melting properties were evaluated by differential scanning calorimetry (DSC) using a modulated differential scanning calorimeter  with a fixed heating rate of 10 °C/min, going from 0 °C to 250 °C for the homopolymer and from 0 °C to 250 °C for the PLLA/PDLA blends. The formation of s-PLA crystalline was evaluated by an X-ray diffractometer (Rigaku D/Max-2500) composed of a Cu The mechanical properties of the PLA blends were evaluated by a Universal Testing Machine Instron 6800 Series) with an adopted method for small size samples [00 SeriesWe expected the utilization of s-PLA crystallites to improve the stereocomplex formation of high molecular weight PLA homopolymers. In this study, we synthesized high molecular weight PLLA and PDLA homopolymers and combined them with s-PLA crystallites. We also compared the effect of s-PLA particle size on high molecular weight s-PLA formation.We started the study by synthesis of high molecular weight PLLA and PDLA homopolymers. Ring-opening polymerization is the most well-known polymerization method that produces PLA from lactide. In this polymerization process, propagation steps were performed by the monomer molecule insert into the metal–oxygen bonds of the catalyst. Catalyst solubility is the key factor for an effective polymerization process in melt . High mow and a broad molecular weight distribution (MWD). High molecular weight was obtained during polymerization due to an effective ratio of the active compound, tin-alkoxide. As reported by Pack et al., the molecular weight of a PLA homopolymer is independent of the initial catalyst (stannous octoate) concentration, but it depends on the tin-alkoxide concentration formed by stannous octoate and 1-dodecanol [The polymerization conversion was calculated using gravimetric analysis see . Polymerodecanol . The broodecanol ,28.m around 177.85 °C. The single peak of Tm shows that there is no stereocomplexation between high molecular weight PLLA/PDLA. The PLLA/PDLA blend with Mw above 1.0 × 105 restricted the formation of s-PLA crystallites [m above 210 °C belongs to s-PLA crystallites. The presence of s-PLA crystallites showed a significant change in the melting behavior of PLLA/PDLA blends under non-isothermal conditions. As polymer blends consist of PLA homocrystallites and s-PLA crystallites, we calculated the total melting enthalpy (∆Hm) as follows [m0 represents the theoretical value of the melting enthalpy of total crystal for PLA homocrystallites and s-PLA crystallites. It was calculated as follows [We investigated the effect of s-PLA crystallites in the high molecular weight PLLA/PDLA blends through the solution casting method. The stereocomplex formation and the crystallinity were evaluated through DSC analysis as tabulated in tallites . High mo follows :(1)ΔHm=Δ follows :(2)ΔHm0=This resulted in tallites . The degtallites . The degtallites :(3)s−PLABased on experimental data, the addition of s-PLA crystallites increased the total crystallinity of the PLLA/PDLA blends concurrently with the degree of s-PLA formation, as shown in The DSC data for s-PLA formation from high molecular weight PLLA/PDLA blends were also confirmed with XRD results, as shown in The increasing total crystallinity and s-PLA degree of PLLA/PDLA blends affect the change in their mechanical properties. The mechanical properties of PLLA/PDLA blends are shown in The stereocomplexation of PLA is one of the more suitable approaches for improving PLA properties. The s-PLA formation from PLLA/PDLA blends was restricted by molecular weight constraints. The presence of s-PLA materials in the high molecular weight PLLA/PDLA homopolymers can initiate the s-PLA formation. The addition of 5% s-PLA particles in high molecular weight PLLA/PDLA blends triggered s-PLA formation up to 49.13% s-PLA degree and 44.34% of the total crystalline fraction. At 10% s-PLA, the formation of s-PLA decreased to 42.75%, but the total crystalline fraction reached 45.07%. The s-PLA particles have capabilities to initiate s-PLA formation and also act as a nucleating agent for the PLA homopolymers. The higher s-PLA degrees and crystalline fraction in the PLLA/PDLA blends aligned with the mechanical property improvements in the PLLA/PDLA blends. The particle size of the s-PLA materials influenced s-PLA formation, total crystalline fraction, and the mechanical properties of the PLLA/PDLA blends. Smaller particle sizes contributed to better particle distribution, as well as more initiation sites for s-PLA formation and nucleation sites for the PLA homopolymer. The s-PLA particles showed the capability to overcome the molecular weight limitation in PLA stereocomplexation.


We observe the expected increase in cell killing following high-LET irradiation that correlates with the increased formation of SCAs as detected by mFISH. Furthermore, we report that cells exposed to low doses of α-particles and 56Fe ions show an enhanced G2-checkpoint response which is mainly regulated by ATR, rather than the coordinated ATM/ATR-dependent regulation observed after exposure to low doses of X-rays. These observations advance our understanding of the mechanisms underpinning high-LET IR effects, and suggest the potential utility for ATR inhibitors in high-LET radiation therapy.The utilization of high linear-energy-transfer (LET) ionizing radiation (IR) modalities is rapidly growing worldwide, causing excitement but also raising concerns, because our understanding of their biological effects is incomplete. Charged particles such as protons and heavy ions have increasing potential in cancer therapy, due to their advantageous physical properties over X-rays (photons), but are also present in the space environment, adding to the health risks of space missions. Therapy improvements and the protection of humans during space travel will benefit from a better understanding of the mechanisms underpinning the biological effects of high-LET IR. There is evidence that high-LET IR induces DNA double-strand breaks (DSBs) of increasing complexity, causing enhanced cell killing, owing, at least partly, to the frequent engagement of a low-fidelity DSB-repair pathway: alternative end-joining , which is known to frequently induce severe structural chromosomal abnormalities (SCAs). Here, we evaluate the radiosensitivity of A549 lung adenocarcinoma cells to X-rays, α-particles and  The year 2021 marks the sixtieth anniversary of the first human-operated spaceflight, which ushered in technological and scientific developments that ignited the exploration and exploitation of space . ActivitThere has been significant progress in understanding the effects of space radiation on the homeostasis of living organisms ,2,3. FurIt has long been known that heavy ions induce a larger proportion of complex structural chromosomal abnormalities (SCAs) than X-rays, from the interaction of more than two DSBs induced in proximity—in one, two or more chromosomes ,12. OtheIn cells of higher eukaryotes, DSBs are mainly repaired by classical non-homologous end-joining (c-NHEJ) and homologous recombination (HR). However, under certain conditions that are presently under intensive investigation, alternative end-joining  and single-strand annealing (SSA) also emerge ,17,18,19The probability of SCA formation is thought to increase with increasing DSB complexity, because it increases with LET. It is also thought to be higher when DSB clusters form, also because it increases with LET ,10. It i2-phase, because cells in this phase of the cell cycle are competent for all available DSB repair pathways [2-phase activate a strong G2-checkpoint that is epistatically regulated by ATM and ATR, i.e., inhibition of either kinase causes its almost-complete abrogation. This mode of regulation changes at higher X-ray doses, with ATM and ATR now regulating the G2-checkpoint both epistatically and independently, i.e., the inhibition of one kinase abrogates the checkpoint only partly and the inhibition of both kinases is required for its complete abrogation [2-phase. Cells irradiated in the S-phase mount when progressing into the G2-phase a checkpoint that is exclusively dependent on ATR, and where the inhibition of ATM prolongs rather than suppresses its activation, suggesting that ATM contributes here to checkpoint recovery [The generation of DSBs by low- or high-LET IR initiates a network of cellular responses, resulting in the activation of DNA damage checkpoints. The activation of DNA damage checkpoints in cells exposed to low- or high-LET IR arrests the normal progression of cells at specific points in the cell cycle to facilitate DSB repair ,25. Partpathways ,27. It irogation . Strikinrecovery . However56Fe ions, by following the radiosensitivity to killing, the formation of SCAs, as well as activation of the G2-checkpoint in cells with suppressed ATM or ATR activity. Our results show, as expected, that A549 and HCT116 cells exhibit increased radiosensitivity to high-LET IR. The reduced cell viability following exposure to α-particles and 56Fe ions correlates with an increased incidence of SCAs that may be a manifestation of increased contribution of low-fidelity alt-EJ. We also analyzed the ATM/ATR-dependent regulation of the G2-phase checkpoint after exposure to low doses of high-LET IR. In contrast to the epistatic regulation of the G2-checkpoint by ATR and ATM following exposure to X-rays [56Fe ions.Continuing previous studies with X-rays ,31, and o X-rays , and in o X-rays , we founATM−, were maintained in McCoy’s 5A growth medium , supplemented with 10% newborn calf serum (Capricorn) and 0.5 μg/mL iron (FeSO4·7H2O), at 37 °C, in an atmosphere of 5% CO2 in air. HCT116 colon cancer cells were grown in McCoy’s 5A growth medium (Sigma-Aldrich), supplemented with 10% fetal calf serum . Exponentially growing cells were subcultured every second day to achieve maximum confluence of about 75%.The human alveolar adenocarcinoma cell line A549, and its ATM-deficient counterpart which is designated here as A459-ATM− cells. For this purpose, A549 cells were transfected with Cas9 expression plasmids, co-expressing GFP, together with a plasmid constructed to express gRNA (5′-ATCATTAAGTACTAGACTCA-3′), targeting exon 2 of the ATM gene. Cell sorting based on GFP expression was utilized to select individual GFP-positive clones, in which the activity of ATM was monitored by immunofluorescence detecting pATM-S1981, a marker of ATM activation; negative clones were selected and expanded for further characterization.CRISPR/Cas9 technology was utilized for the generation of A549-All inhibitors were dissolved in dimethyl sulfoxide (DMSO) and were added to the culture medium one hour before irradiation. The ATR inhibitor, VE-821 , referred to as ATRi, was applied at a concentration of 5 μM, whereas the ATM inhibitor KU55933 (ATMi),  was applied at a concentration of 10 μM. In experiments measuring cell survival by colony formation as endpoints, cells were transferred to inhibitor-free growth medium 24 h after irradiation.Irradiations with low-LET X-rays  were carried out in Essen using an X-ray machine . Tube voltage and current were set to 320 kV and 10 mA, respectively, and a 1.65 mm aluminum filter  was used to absorb “soft” X-rays. The dose rate was ~3.4 Gy/min and was determined using an in-field ionization monitor calibrated with a PTB dosimeter . The radiation dose was confirmed with Fricke’s chemical dosimetry. An even dose distribution within the irradiation field was achieved by rotating the radiation table. Cells were irradiated at a distance of 500 mm. Immediately after irradiation, cells were returned to the incubator.2 tissue culture flasks and were incubated for at least 24 h at 37 °C in Essen. The following day, cells were transported in an insulated container filled with warm pads and allowing active heating to maintain the temperature of the cells close to 37 °C. Upon arrival at the GSI, cells were promptly incubated at 37 °C under standard growth conditions, and when possible, were allowed to recover for several hours from the transportation stress. Cells were exposed to 1 GeV/amu 56Fe ions, LET = 150 keV/μm. Dosimetry was carried out with a calibrated farmer chamber . The absolute particle fluence was measured with a calibrated ionization chamber  at the beam exit window, and the homogeneity of the scanned field was regularly checked using radiochromic EBT films . LET was calculated using “ATIMA”, a program developed at the GSI. After radiation exposure, cells were immediately transported to Essen, while maintaining them close to 37 °C. When necessary, during transportation, samples were transferred to ice to measure the kinetics of responses under investigation. The limited availability of HI for biological experiments compromised the number of repeats that could be performed.Exposure to heavy ions (HIs) was carried out at the GSI (Helmholtzzentrum für Schwerionenforschung GmbH) in Darmstadt, Germany. Typically, cells were seeded in 25 cm241Am α-particle source was plated as an Am2O3 compound  into a silver foil and was covered with a gold layer of 2 μm in thickness. An aluminum ring with inner diameter of 50 mm was used to delimit the source active area. For α-particle exposures, sterile glass rings (inner diameter of 45 mm and height of 22 mm) with a 1.5 μm thick Mylar foil bottom were used as previously described [4He ions generated from the 241Am source. During the irradiation, the source chamber was flushed with helium while the target chamber contained normal air. A homogenous dose distribution was achieved by rotating the α-particle source and by wobbling the collimator. The dose rate was 1.32 Gy/min, and the mean LET was 124 keV/μm [2 concentration) as cells subjected to radiation.The device for α-particle irradiation has previously been described in detail , and conescribed . Cells w4 keV/μm . Non-irrATM− cells following the exposure to IR of different LET, cells were plated in triplicate four hours after exposure to X-rays and α-particles, or immediately upon arrival in Essen, which was typically four to five hours after irradiation with 56Fe ions. Cells were grown for 11 days and stained with 1% crystal violet dissolved in 70% ethanol. Colonies were counted using a low-magnification binocular microscope. Alternatively, dishes were scanned, and the colonies were scored on the digitized images.To assess the colony-forming ability of A549, HCT116 and A459-56Fe ions irradiation, where only one experiment could be carried out.To score SCAs, mFISH analysis was employed 48 h after irradiation. To accumulate cells at metaphase, colcemid  was added over 2–3 h at a concentration of 0.1 μg/mL. Cells were harvested by trypsinization and incubated for 15 min in 10 mL hypotonic solution . Subsequently, cells were fixed in 10 mL ice-cold fixative—3:1 methanol :acetic acid  and kept at 4 °C overnight. After several washes with the fixative, metaphase spreads were prepared and dried for 24 h at room temperature. mFISH was performed using a 24 × Cyte Multicolor FISH probe for human chromosomes , according to the manufacturer’s protocol. An automated imaging system  was used to obtain high-quality images of metaphase chromosomes. For the metaphase search, the M-Search module of the Metafer software using the 10× air objective of a Zeiss microscope  was used. Metaphases were captured at a magnification of 63×, using the AutoCapt setting of the Metafer software. Images were analyzed using Isis Software. For analysis, at least 50 metaphases were scored in each of three independent repeats of each experiment—except for 6 cells were fixed in 70% ice-cold ethanol and were permeabilized for 15 min in ice-cold PBS supplemented with 0.25% Triton X-100. Cell pellets were incubated in 0.05% Tween-20, 1% BSA in PBS for 45 min at RT, followed by incubation with primary rabbit anti-H3-pS10 specific antibody  for 2 h at RT. Cells were washed three times with PBS and incubated in AlexaFluor 488-conjugated goat-anti rabbit-IgG . Finally, DNA was stained with PI for 30 min at 37 °C. All incubation steps were performed under gentle agitation. Analysis was carried out in a Gallios flow cytometer  by measuring 2 × 104 cells per sample; proper gating was applied to select H3-pS10-positive events that represented mitotic cells. The mitotic index (MI) was determined as the fraction of cells in mitosis and is shown normalized to the MI of non-irradiated controls. The actual MIs of the controls that were used for normalization are given in the legends of the corresponding figures.Two-parameter flow cytometry was employed to simultaneously measure DNA content by propidium iodide (PI) staining and mitotic cells by quantification of the phosphorylated histone H3 at Serine 10 (H3-pS10). Briefly, 0.6–1.0 × 10Auto-phosphorylation of ATM at S1981 is a marker for ATM activation. Therefore, to detect ATM deficiency in selected A549 clones after CRISPR/Cas9 treatment, indirect immunofluorescence was applied to visualize pATM-S1981 foci in cells exposed to X-rays, as described elsewhere . Briefly56Fe ions exhibited increased radiosensitivity compared to X-rays . Moreover, we also studied the impact of ATMi and ATRi on SCA formation in HCT116 cells after exposure to X-rays or α-particles. To compare the effect of ATMi and ATRi at the cell survival level with their impact on SCA formation, we carried out chromosome analysis as described above c,d. FiguIt is thought that the exposure of cells to high-LET IR increases cell killing over that generated by low-LET IR by altering the rules of DSB repair pathway engagement ,9,34. ThOur results show an LET-dependent increase in SCAs in both A549 and HCT116 cells, which are considered hallmarks for alt-EJ engagement and are unlikely to be generated by HR. SCAs can be lethal or carcinogenic events, and therefore the engagement of repair pathways favoring their formation, such as alt-EJ, must be carefully balanced by the cell. A clear net benefit must arise for genomic stability to justify the recruitment of alt-EJ. We assume therefore that following exposure to high-LET IR, forms of DSBs are generated that engage first-line DSB repair pathways such as HR or c-NHEJ. However, when these repair pathways are challenged and occasionally ultimately fail owing to the complexity of the DSB, alt-EJ becomes the last resort—a form of backup. The choice for the cell is therefore to either utilize alt-EJ despite the associated risks, or to leave the DSB unprocessed. It is likely that the degree of genomic preservation ensured by alt-EJ engagement is higher than that associated with the option of leaving the DSB unprocessed and risk the loss of genomic material. Notably, such decisions are likely required for only a small fraction of DSBs that are actually ultimately shunted to alt-EJ. In addition, the majority of alt-EJ events will rejoin the correct ends and will not lead to SCAs. Thus, infrequent utilization, as a last resort scenario, may underpin the evolutionary appearance and preservation of alt-EJ.2-checkpoint in cells exposed to low doses of low-LET IR in the G2-phase. Indeed, we were able to demonstrate that under these conditions, ATM and ATR operate in the form of a functional module within which the two kinases epistatically regulate G2-checkpoint activation [We have recently demonstrated intriguing contributions and crosstalk between ATM and ATR in the regulation of the Gtivation ,29. Our tivation ,29. The 2-checkpoint has been reported before and after the exposure of cells to carbon ions [56Fe ions are in general agreement with the findings of that report. Another report showed that ATR inhibition abrogates the G2-checkpoint, induces micronuclei, and causes cell radiosensitization after exposure to carbon ions [A similar dominant role of ATR in the Gbon ions , and ourbon ions : all fin2-checkpoint after high-LET IR suggests the engagement of DSB repair pathways utilizing DNA-end resection and therefore generating the ssDNA required to activate ATR. Alt-EJ falls in this category of DSB repair pathways. DNA-end resection exposes microhomologies and larger regions of homology that support alt-EJ, and, if long enough, SSA as well [The increased functional involvement of ATR in the G as well . SSA is  as well . FurtherMLH1 or MSH2 deficiency of HCT116 cells [HCT116 cells are more sensitive to X-rays than A549 cells. This may reflect the documented 16 cells , as well16 cells ,43. Our Our observations are also in line with previous studies showing that the transient inhibition of ATM causes the accumulation of persistent chromosome aberrations ,45. Inhi1-checkpoint, in contrast to most NSCLC cells, it is proposed that selective tumor radiosensitization can be achieved by abrogation of the G2-checkpoint using inhibitors of ATM or ATR [2-checkpoint documented in our study, as well as in previous studies, after exposure to high-LET IR should be helpful in the goals of this platform, when high-LET IR modalities become integrated as treatment options.Recently, ATM and ATR have been selected as potential pharmacological targets in the CONCORDE platform—the first phase Ib randomized, open-label, multi-institution, multi-arm clinical trial seeking to determine the safety profile of several DDR inhibitory agents in combination with fixed-dose radical radiotherapy in locally advanced non-small cell lung carcinomas (LA-NSCLC). Non-malignant cells possess a functional GM or ATR . The alt2-checkpoint points to mechanistic shifts in the crosstalk between ATM and ATR in the regulation of this endpoint and emphasizes the increased role of DNA end-resection in DSB repair and the potential of ATR inhibitors in the clinical setting. Collectively, this information is relevant to the utilization of high-LET IR in cancer therapy and to radiation protection during space travel.The present study has confirmed the enhanced cellular radiosensitivity following exposure to high-LET IR and reinforces its correlation with the increased incidence of SCAs that, in turn, implies the increased engagement of error-prone DSB repair pathways. The prevailing role of ATR in the regulation of G


And terpinen-4-ol at 0.32% could kill all bacteria and clear all biofilms. Untargeted metabolomic and transcriptomic analyses showed that terpinen-4-ol strongly inhibited DNA and RNA biosynthesis in MRSA at 2 h after treatment by affecting genes and metabolites related to purine and pyrimidine metabolic pathways. Some differential genes which play important roles in DNA synthesis and the production of eDNA from biofilm exposed to terpinen-4-ol was also significantly decreased compared with that of the control.We observed that terpinen-4-ol has good antibacterial activity and inhibits the formation of MRSA biofilm. The MIC and MBC values for terpinen-4-ol against Terpinen-4-ol has good antibacterial activity and significantly inhibits the formation of MRSA biofilm by inhibiting purine and pyrimidine metabolism. Melaleuca alterniflora in Australia by steam distillation, with the smell of camphor and cooling sensation of menthol [In recent years, essential oils have become widely used in the biomedical, cosmetics and food industries and other fields due to their broad antibacterial activity and few side effects . Numerou menthol . TTO is  menthol .Terpinen-4-ol was considered to be the main active ingredient of TTO , and ISOStaphylococcus aureus (S. aureus) is one of the earliest described pathogens in humans and one of the most common causes of infection [S. aureus has become increasingly serious and has led to the MRSA pandemic [nfection . Due to pandemic . MRSA capandemic . Biofilmpandemic . The bacpandemic . Currentpandemic . Antibiopandemic  .S. aureus were 0.08% ~ 0.32%  and 31.20% ± 2.13% (P < 0.01). It can be seen that higher concentrations of terpinen-4-ol effectively inhibited the formation of biofilm.The antibiofilm activity of terpinen-4-ol was also assessed by the crystal violet staining. As shown in Fig. Streptococcus mutans, and Lactobacillus acidophilus [Porphyromonas gingivalis, Fusobacterium nucleatum [In this study, terpinen-4-ol behaved its inhibition in MRSA biofilm formation. It also confirmed other authors’ results that terpinen-4-ol could destroy the biofilm of many oral pathogens such as, dophilus  Porphyroucleatum . We coulP < 0.01) for MRSA mature biofilm. As the concentration of terpinen-4-ol decreased, the clearance rate gradually decreased. The viability of the preformed MRSA biofilm exposed to terpinen-4-ol was also verified by CLSM analysis. As shown in Fig. The destruction of biofilms by terpinen-4-ol was also evaluated. As shown in Fig. Terpinen-4-ol showed its activity in clearing MRSA biofilm. Generally, it is more difficult to remove mature biofilm than to inhibit its formation. Thus, terpinen-4-ol could consider to be a potential antibiofilm agent, for it can not only inhibit the formation of biofilms, but also destroy mature biofilm. Therefore, it is necessary to further study the anti-biofilm activity of terpinene-4-ol and explain its mechanism of action.To understand the changes in MRSA due to treatment with terpinen-4-ol, we measured the transcriptome of MRSA exposed to terpinen-4-ol and screened 304 DEGs as defined by FC > 2 and Q < 0.05. Among these DEGs, 159 genes were downregulated and 145 genes were upregulated in terpinen-4-ol-treated relative to control  in terpinen-4-ol-treated group. Some of those pathways related to the formation of MRSA biofilm. For example, BCAAs, synthesized by 2-oxocarboxylic acid, regulate the biosynthesis of bacterial amino acids [KEGG pathway analysis was used to obtain insights into the biological functions of the DEGs Fig. . Compareno acids  and nuclno acids  by combino acids –28. TherFurthermore, there were other pathways that warrant our attention, such as purine metabolism, quorum sensing (QS), and β-Lactam resistance. Most of these pathways are related to the synthesis of nucleic acids or ATP, which are important to biofilm formation of MRSA. We then used metabolomics to verify the results of the transcriptomics analysis.P < 0.05, and VIP value > 1.0. Among these differential metabolites, 125 were upregulated and 90 were downregulated in terpinen-4-ol-treated MRSA relative to control MRSA . Caffeine can be converted to purine, and we found that many intermediate products of purine metabolism and the contents of its constituents have decreased, including xanthine and xanthosin, which are intermediate products of purine metabolism, guanosine, and adenosine, which are components of purine metabolism, 2′-deoxyadenosine, and deoxyadenosine, which are components of DNA.Twelve sets of data from the six biological replicates were analyzed to determine the changes in metabolites that occur in MRSA following treatment with terpinen-4-ol. PCA analysis Fig.  showed tRSA Fig. . EighteeRSA Fig.  were ideIn addition, there were also some metabolites related to pyrimidine metabolism have decreased. The content of these substances is closely related to DNA synthesis, and the reduction of their content may lead to a reduction in the amount of bacterial DNA. Thus, we conjectured that terpinen-4-ol achieves its anti-MRSA activity by inhibiting the synthesis of DNA.eDNA an important component of the EPS released by the lysis of some bacteria during the formation of the biofilm , and it deoD was upregulated by 12.378 ± 0.541 times, and the expression of pyrB was downregulated by 3.049 ± 0.147 times. Those results were largely consistent with the transcriptome results that terpinen-4-ol inhibit purine and pyrimidine metabolism.To verify our results, we also performed qRT-PCR verification of some of the differential genes, which play important roles in DNA synthesis Table . In terpnrdF, arcC, deoD, pyrF, pyrB and carB. In the metabolome results, the contents of 11 metabolites related to nucleic acid synthesis, including xanthine, 2′-deoxyadenosine, inosine, cytosine, thymidine, and deoxyadenosine, were significantly reduced in terpinen-4-ol-treated MRSA relative to control MRSA.The differential genes identified from the transcriptomics analysis and the differential metabolites identified from metabolomics analysis were represented in the KEGG pathway database Fig. . After tATP is composed of adenine, ribose and 3 phosphate molecules, and is the most direct source of energy in living organisms . The redpyrB and carB are the key enzymes of the nucleotide rescue pathway, being very important for the rescue synthesis of nucleotides. In our research, the expression of these two genes was inhibited by terpinen-4-ol treatment. Furthermore, the levels of four nucleotides, the raw material for UMP synthesis, declined significantly after terpinen-4-ol treatment, indicating that the salvage synthesis of pyrimidine nucleotides was inhibited by terpinen-4-ol. The inhibition of pyrimidine nucleotides might block the production of modified nucleotides acting as signal molecules for biofilm formation, such as c-di-GMP [The biosynthesis of pyrimidine nucleotides can affect biofilm by influencing the expression of the genes csgDEFG . The intc-di-GMP , which iTerpinen-4-ol has good antibacterial activity and significantly inhibits the formation of MRSA biofilm. It demonstrated the excellent potential in anti-biofilm drug. The results of the transcriptomics and metabolomics analyses suggest that terpinen-4-ol inhibits the growth of MRSA by inhibiting purine and pyrimidine metabolism. According to the decrease of eDNA in MRSA biofilm, it is further speculated that the mechanism of terpinen-4-ol inhibiting the formation of MRSA biofilm may also inhibit the synthesis of purines and pyrimidines. These findings provide new insights into the antibacterial mechanisms of terpinen-4-ol and may promote the study of the pharmacological activity of terpinen-4-ol as a natural antibacterial product.S. aureus were obtained from the manure of the dairy farm in Gansu China, and were labeled S-1 through S-12. MHA and MHB were obtained from Aobox Biotechnology . TSB and BHI were obtained from Oxoid , and terpinen-4-ol was obtained from Sigma Aldrich .MRSA (ATCC 43300) was obtained from Beina Biotechnology . Twelve isolates of S. aureus were tested in this study.The MIC of terpinen-4-ol was determined according to the Clinical and Laboratory Standard Institute (CLSI) guidelines. One standard strain and 12 clinical strains of Time-kill curve of terpinen-4-ol against MRSA strain ATCC 43300 was generated. MRSA was cultivated in MHB with terpinen-4-ol and incubated at 37 °C. The number of MRSA was measured by the plate counting method at regular time intervals.The adhesion of biofilm was measured by crystal violet staining and CLSM. The crystal violet staining method was performed according to a previous report and used to determine the inhibition and clearance of MRSA biofilm by terpinen-4-ol at different concentrations. For the effect of terpinen-4-ol on the removal of biofilms, a mature biofilm is first cultivated, and then different concentrations of terpinen-4-ol was added. After a certain period of time, the amount of biofilm is determined by crystal violet. The biofilm was cultured and measured following Srdjan Stepanović’s report .CLSM was performed by staining a pre-established biofilm with the LIVE/DEAD BacLight Bacterial Viability kit  according to the manufacturer’s instructions. CLSM images were captured using an LSM 800  with a 40× objective lens. ZEN 2.3 was used to analysis and exporting images of CLSM.MRSA was exposed to terpinen-4-ol at a concentration of 1/2 × MIC  for 2 h, and 3 treatment samples and 3 control samples were analyzed. Total RNA was extracted from MRSA using an RNAprep Pure Bacteria Kit . The transcriptome sequencing and analysis were conducted by Novogene  using the Illumina sequencing platform (Illumina NovaSeq). DEGs screening and KEGG enrichment analysis respectively used Bowtie v2.2.3 and KOBAS.P value represents the contribution rate of the difference in metabolites in different groups; the Fold Change (FC) is for each metabolism The ratio of the mean value of the repeated quantitative values of all organisms in the comparison group; combined with the P value of T-test to find the differentially expressed metabolites, set the threshold value to VIP > 1.0, and the multiple of difference FC > 1.5 or FC < 0.667 and P value < 0.05, screen out the different metabolites.MRSA was exposed to terpinen-4-ol at a concentration of 1/2 × MIC  for 2 h, and 6 treatment samples and 6 control samples were analyzed. MRSA (100 mg) were individually grounded with liquid nitrogen and the homogenate was resuspended with prechilled 80% methanol and 0.1% formic acid by well vortexing. Take an equal volume sample from each experimental sample and mix it as a quality control sample for quality control to ensure the quality of the final collected data. And the metabolites analysis was carried out to analyze using a Vanquish UHPLC system coupled with an Orbitrap Q Exactive series mass spectrometer . The raw data files generated by UHPLC-MS/MS were processed using the Compound Discoverer 3.1  to perform peak alignment, peak picking, and uantitation for each metabolite. MetaX software  was used600 was measured by enzyme-labeled instrument, and the eDNA was extracted. The concentration of eDNA was measured with an DNA/Protein Analyzer . The relative content of eDNA was measured in terms of eDNA content per unit of biofilm.The extraction of eDNA in MRSA was conducted as previously reported . First, The extraction of total RNA was performed using the Simply P Total RNA Extraction Kit . The cDNAs were synthesized by the PrimeScript™ RT reagent Kit with gDNA Eraser  . The quantitative real-time polymerase chain reaction (qRT-PCR) was conducted using QuantStudio  with TB Green® Premix Ex Taq™ II . The primers and genes are listed in Table P < 0.05 was considered to be significant.All data except RNA-seq and metabolomics were reported as means ± SD. The significance of differences was determined by t-test using SPSS 23.0 (SPSS Inc.). A value of


Transferring results obtained in cardiovascular outcome trials (CVOTs) to the real-world setting is challenging. We herein transposed CVOT results to the population of patients with type 2 diabetes (T2D) seen in routine clinical practice and who may receive the medications tested in CVOTs.We implemented the post-stratification approach based on aggregate data of CVOTs and individual data of a target population of diabetic outpatients. We used stratum-specific estimates available from CVOTs to calculate expected effect size for the target population by weighting the average of the stratum-specific treatment effects according to proportions of a given characteristic in the target population. Data are presented as hazard ratio (HR) and 95% confidence intervals.Compared to the target population , the CVOT population  was younger and had a two to threefold greater prevalence of cardiovascular disease. EMPA-REG was the CVOT with the largest variety of details on stratum-specific effects, followed by TECOS, whereas DECLARE and PIONEER-6 had more limited stratum-specific information. The post-stratification HR estimate for 3 point major adverse cardiovascular event (MACE) based on EMPA-REG was 0.88 (0.74–1.03) in the target population, compared to 0.86 (0.74–0.99) in the trial. The HR estimate based on LEADER was 0.88 (0.77–0.99) in the target population compared to 0.87 (0.78–0.97) in the trial. Consistent results were obtained for SUSTAIN-6, EXSCEL, PIONEER-6 and DECLARE. The effect of DPP-4 inhibitors observed in CVOTs remained neutral in the target population.Based on CVOT stratum-specific effects, cardiovascular protective actions of glucose lowering medications tested in CVOTs are transferrable to a much different real-world population of patients with T2D.The online version contains supplementary material available at 10.1186/s12933-021-01300-y. Prevention of cardiovascular disease is a major objective of diabetes care . In the We herein used an innovative approach to transpose the effects of GLP-1RA or SGLT2i observed in the respective CVOTs to a large unselected target population of patients who were followed under routine specialist care and could potentially be prescribed such medications.Since the method for transposing trial effects to the target population relies on the availability of stratum-specific information of effect , we seleIn Italy, GLP-1RA and SGLT2i can be prescribed only by diabetes specialists. Therefore, as a target population of individuals with T2D who could receive such medications in real-life, we used the DARWIN-T2D  database . DARWIN-The method for transposition is escribed as a flow-chart in Fig. As a simple example, let us consider the “gender” variable in the REWIND trial, where 46% of participants were women. In the target population, 44% of subjects were women. In REWIND, the stratum-specific HR estimates were 0.85 (95% C.I. 0.71–1.02) for females and 0.90 (95% C.I. 0.79–1.04) for males. Then, the weighted HR estimate for the variable “gender” is computed by the formula:The standard deviations across strata were pooled to obtain the 95% confidence interval. The calculation was performed for one characteristic at a time. Then, the unweighted average of the estimated transposed treatment effect of each characteristic was used to summarize the post-stratification estimates of treatment effect. Analyses were performed using R version 3.5.2 .The analysis was conducted only for CVOTs having usable data for transposition. For example, transposition could not be performed for the CANVAS study because reporting of stratum-specific effects was not accompanied by numbers of patients in each stratum . The anaThe number of variables that defined stratum-specific effects ranged from a maximum of 28 for EMPA-REG . TherefoAfter excluding patients with missing data of key variables, the target population of the DARWIN-T2D study was composed of a total of 139,726 patients, but not all information was available for all patients. Table After transposition to the target population, the estimated HR was significantly lower than 1.0 for LEADER , SUSTAINThe effect on 3p-MACE observed in EXSCEL , PIONEERDespite major differences between patients with T2D enrolled in CVOTs and patients with T2D seen in routine clinical practice –14, our Due to the specific population of patients enrolled in CVOTs, doubts have been cast on whether significant protective effects observed for some of the active drugs being tested could be transferred to the entire population of patients who could receive such medications in clinical practice . In mostTo the best of our knowledge, there is no prior attempt to transpose effects of diabetes medications observed in CVOTs to a real-world population, with quantitative estimates. Thus, our new findings can help reducing inertia in the use of GLM for which solid cardiorenal protective data exist .The post-stratification method described by Hong et al. can be used to generalize trial results without accessing individual-level data . The golIt is important to note that most CVOTs reporting superiority of active drugs versus placebo for cardiovascular protection was confirmed after transposition to the real-world population. This is the case for LEADER, SUSTAIN-6, REWIND, and DECLARE (second co-primary endpoint), while it was not for EMPA-REG Outcome. The reasons why the significantly lower rate of 3P-MACE among patients randomized to empagliflozin in the EMPA-REG Outcome trial was not significant after transposition to the target population can be manifold. These include the presence of nominally significant heterogeneity observed in subgroups of patients divided by age and baseline HbA1c , the 2:1It is important to note that only 30% to 50% of patients enrolled in CVOTs were recruited in Europe, questioning generalizability of the findings to European populations. We also would like to note that the target population addressed in this study might not be representative of patients with T2D in other countries. We included only patients followed at specialist outpatient clinics because, in Italy, only diabetes specialists but not general practitioners, can prescribe GLP-1RA, SGLT2i and DPP-4i . TherefoNotwithstanding the above-described limitations, we herein provide the first estimate that cardiovascular protection by diabetes drugs investigated in CVOTs could apply to a very different and highly heterogeneous population of patients with T2D seen in routine care.Additional file 1:Table S1. REWIND. Table S2. SUSTAIN-6. Table S3. DECLARE HHF/CVD. Table S4. DECLARE MACE. Table S5. EMPA-REG. Table S6. LEADER. Table S7. PIONEER-6. Table S8. TECOS. Table S9. SAVOR-TIMI. Table S10. EXCEL.


Antigen testing for SARS-CoV-2 (AGT) is generally considered inferior to RT-PCR testing in terms of sensitivity. However, little is known about the infectiousness of RT-PCR positive patients who pass undetected by AGT. In a screening setting for mildly symptomatic or asymptomatic patients with high COVID-19 prevalence (30–40%), 1141 patients were tested using one of five AGTs and RT-PCR. Where the results differed, virus viability in the samples was tested on cell culture (CV-1 cells). The test battery included AGTs by JOYSBIO, Assure Tech, SD Biosensor, VivaChek Biotech and NDFOS. Sensitivities of the ATGs compared to RT-PCR ranged from 42% to 76%. The best test yielded a 76% sensitivity, 97% specificity, 92% positive, and 89% negative predictive values, respectively. However, in the best performing ATG tests, almost 90% of samples with “false negative” AGT results contained no viable virus. Corrected on the virus viability, sensitivities grew to 81–97% and, with one exception, the tests yielded high specificities >96%. Performance characteristics of the best test after adjustment were 96% sensitivity, 97% specificity, 92% positive, and 99% negative predictive values . We, therefore, believe that virus viability should be considered when assessing the AGT performance. Also, our results indicate that a well-performing antigen test could in a high-prevalence setting serve as an excellent tool for identifying patients shedding viable virus. We also propose that the high proportion of RT-PCR-positive samples containing no viable virus in the group of “false negatives” of the antigen test should be further investigated with the aim of possibly preventing needless isolation of such patients. RT-PCR testing is currently considered the most reliable technique for identification of patients infected with the new coronavirus SARS-CoV-2. However, antigen tests from nasopharyngeal swabs (AGTs) providing rapid response at the point-of-care are also deemed suitable for screening purposes . In a scHowever, few studies take into account the viability of the virus detected by RT-PCR, which is associated with infectiousness  ,8,9. We The study was approved by the local Ethics Committee, No. NsPKar/19956/2020/SEK. All patients signed informed consent prior to inclusion in the study.The tests were compared in a real-world high-throughput setting of asymptomatic (48.3%) and/or mildly symptomatic (51.7%) patients. Consecutive individuals referred to our testing point due to a contact with a SARS-CoV-2 positive person or due to having COVID-19-like symptoms with course not requiring hospitalization were included in the study. Two (one from each nostril) nasopharyngeal swabs were taken from each patient by trained medical personnel. One swab was placed into the transport medium , the other into the medium supplied with the respective AGT (one AGT per person was performed). The AGT was performed immediately, while RT-PCR and, if necessary, viability tests were performed after the sample was transported into the laboratory. The tests were performed within 24 h of sampling; where this was not possible, samples were frozen at −80 °C and thawed immediately before testing.Five antigen tests, namely SARS-CoV-2 Antigen Rapid Test Kit (JOYSBIO (Tianjin) Biotechnology Co., Ltd., Tianjin, China); Ecotest COVID-19 Antigen Rapid Test ; Standard Q COVID-19 Ag ; Immupass VivaDiag™ SARS-CoV-2 Ag Rapid Test (VivaChek Biotech (Hangzhou) Co. Ltd., Hangzhou, China); and ND COVID-19 Ag test  were provided to us for evaluation by the Ministry of Health of the Czech Republic. Testing was performed according to the manufacturers’ instructions.6 RNA copies/mL sample was detected at Ct 26. By calculation, the Ct cut-offs for individual groups are approximately 1.28 × 108 (Ct = 20) and 1.25 × 105 (Ct = 30) RNA copies/mL.RT-PCR analysis was performed using the Automated RNA Isolation Kit for Agilent Bravo  and the PCR detection kit COVID-19 Multiplex RT-PCR Kit  according to the manufacturer’s instructions. The isolation procedure resulted in an approx. 3× increase in RNA concentration. The detection was based on the proof of two SARS-CoV-2 genes, namely genes coding the spike protein and endoRNAse. The entire process was controlled through isolation and amplification of a synthetic internal control. Cycle thresholds (Ct) for both genes were recorded and the mean Ct from each sample was included in the analysis. Samples were evaluated as Strongly positive (Ct ≤ 20), Positive (20 < Ct ≤ 30), Weakly positive (30 < Ct ≤ 40), and Negative (CT > 40). The reference standard supplied by the PCR kit manufacturer containing 2 × 10Virus viability was assessed only in samples where the result differed between the antigen test and RT-PCR. Testing was performed in monolayer CV-1 cells (African green monkey kidney fibroblasts) cultured at 37 °C in the E-MEM medium  in Leighton tubes. Cells were inoculated with 300 µl of the sample used for the RT-PCR testing or blanks. The cultures were examined daily under the microscope (100–200× magnification) for changes indicating a cytopathic effect of the virus. After 7 days (or sooner if the cytopathic effect was observed in approx. 75% of cells), the cells were passaged (1:6) and cultured for an additional 7 days. If no cytopathic effect (virus action) was observed over the next 7 days, the sample was declared free of viable virus. Where cytopathic effect was observed, SARS-CoV-2 presence was verified by RT-PCR.11 RNA copies/mL), both directly and after freezing at −80 °C. The cytopathic effect was observed from approx. 104 RNA copies/mL, which, with the infectious particles:RNA ratio in the tissues ranging typically between 1:1000 and 1:10,000 [The sensitivity of the method was verified through serial dilution of the virus stock suspension prepared by cultivation and measured by PCR (3 × 10https://www.stata.com/stata14/) and Clinical Calculator 1 (http://vassarstats.net/clin1.html) comparing the AGT results to the result of a) RT-PCR only and b) RT-PCR corrected on the viability in discrepant samples . 95% confidence intervals were calculated for all parameters. The dataset is available from Figshare at https://doi.org/10.6084/m9.figshare.14096648.v1.Antigen test parameters  were calculated in Stata v.14  were SARS-CoV-2 negative according to RT-PCR, 193 samples (16.9%) were weakly positive (30 < Ct ≤ 40), 196 (17.2%) positive (20 < Ct ≤ 30), and 8 (0.7%) strongly positive (Ct ≤ 20). Most discrepant results between AGTs and RT-PCR were detected, as expected, in the group of patients with weak RT-PCR positivity (81.4% of discrepant results). All patients in the patient group with strong RT-PCR positivity were detected also by the AGTs. More details are shown in the Performance parameters of individual tests in combination with the prevalence of SARS-CoV-2 positive samples in the screening setting are detailed in In this study, we have compared five antigen tests (AGTs) for detection of SARS-CoV-2 in nasopharyngeal swabs and where the results of RT-PCR and AGT disagreed, we performed viability testing. As far as we know, the presented study is the largest one evaluating the virus viability in association with the antigen testing in a real-world high-prevalence setting so far. As expected, the highest number of discrepant results was in the group of patients with high Ct numbers (weak positivity), with approx. 70% of all discrepancies falling within these groups. The only exception was the ND COVID test with a very high false-positive rate of 44%.Relative to RT-PCR only, all tests performed significantly poorer in our study than reported by the manufacturers. This is true even for Standard Q, verified by multiple studies . HoweverRegardless of the performance of the individual tests, the most important outcome of our study is the generally very low rate of viable virus presence in the samples which are conventionally considered to be “false negatives of the antigen tests,” which was particularly true for the samples with weakly positive RT-PCR results  would bLastly, and most importantly, we cannot be 100% sure that patients without a viable virus in the nasopharynx do not shed viable virus from the throat or other parts of the upper respiratory tract . Nevertheless, Wölfel et al.  found noWell-performing AGTs could also possibly serve for termination of isolation in patients with SARS-CoV-2. It is well-known that a week to 8 days after the onset of symptoms, no virus isolation was successful in patients with mild course of the disease ,13,16, aLimitations of this study include the uneven number of patients tested by individual AGTs and, in particular, the low number of samples tested using the Standard Q test. This was, however, given by the numbers of tests provided to us by the Ministry of Health. Another limitation was that only nasopharyngeal swabs were performed. However, we opted for using the same technique that was used for the collection of samples for RT-PCR to be able to directly compare these two methods. Lastly, we did not perform viability testing in all samples but only in the discrepant ones. We concede that knowing the virus viability in all samples would be a great help for understanding the performance of AGT and RT-PCR; however, performing viability testing in more than 1100 samples was just not practically feasible. Still, we believe that such a study  should be performed and is already long overdue.Our results show that, when using suitable antigen tests, viable virus was present only in approx. 12% of the antigen test “false negatives” (when compared to RT-PCR only). Correcting on that, the best of the evaluated tests yielded 96.4% sensitivity, 97% specificity, and perhaps most importantly, 98.7% negative predictive value. This leads us to propose that (a) virus viability testing, at least of the discrepant samples, should form a part of AGT evaluation and validation process; (b) we propose that antigen testing using a well-performing AGT could represent a valid tool for infection control in high prevalence settings, and (c) that more detailed research on the patients who are “missed” by (well-performing) AGT is needed with respect to their infectiousness to determine whether or not they pose a threat from the perspective of disease spreading.


Due to the simultaneous detection capability, the ICP-ToF-MS might, however, be more favorable when several isotopic systems are analyzed within one measurement. In a case study, the GC/ICP-ToF-MS coupling was applied for analysis of MMHg in sediments of Finow Canal, a historic German canal heavily polluted with mercury. Mass fractions between 0.180 and 41 μg/kg (w(Hg)MMHg) for MMHg, and 0.056 and 126 mg/kg (w(Hg)total) for total mercury were found in sediment samples taken from the canal upstream and downstream of a former chemical plant.A recently introduced inductively coupled plasma-time-of-flight-mass spectrometer (ICP-ToF-MS) shows enhanced sensitivity compared to previous developments and superior isotope ratio precision compared to other ToF and commonly used single-collector ICP-MS instruments. Following this fact, an improvement for isotope dilution ICP-MS using the new instrumentation has been reported. This study aimed at investigating whether this improvement also meets the requirements of species-specific isotope dilution using GC/ICP-MS, where short transient signals are recorded. The results of the analysis of monomethylmercury (MMHg) of a sediment reference material show that isotope ratio precision of ICP-MS instruments equipped with quadrupole, sector-field, and time-of-flight mass analyzers is similar within a broad range of peak signal-to-noise ratio when analyzing one isotopic system. The procedural limit of quantification (LOQ) for MMHg, expressed as mass fraction of Hg being present as MMHg, The online version contains supplementary material available at 10.1007/s00216-021-03497-z. Isotrecision , 5. Howerecision .srel) of 0.2–0.5% using high analyte concentrations [These hyphenations of ICP-MS, especially using HPLC and GC, have substantial advantages in analyzing (trace) elemental species, including high sensitivity and selectivity , 8. Moretrations , 19. For0) and organic species, such as MMHg [An area of broad interest is Hg speciation because of its complex biogeochemical cycle , includi as MMHg –22, alth as MMHg , 23. Nat as MMHg , 22, cor as MMHg . MMHg ha as MMHg  as well  as MMHg  and bird as MMHg  living i as MMHg .One example of a highly Hg-contaminated waterbody is Finow Canal, the oldest artificial waterway still in operation in Germany, with Hg mass fractions of up to or higher than 100 mg/kg in the sediment , 28, whiIn this study, we evaluate the performance of a recently commercialized ICP-ToF-MS in SSID GC/ICP-MS and compare it with widely used ICP-Q-MS and ICP-SF-MS instruments. The practicability of the ICP-ToF-MS is shown by a case study conducting mercury speciation analysis in sediments of Finow Canal on length from before the known polluted sited into the confluence with Oder-Havel Canal.Samples were taken from eight locations following the Finow Canal  downstream from the crossing of Finow Canal and Oder-Havel Canal near Marienwerder to the confluence of both canals near the town Niederfinow Fig. . Two addThe samples were freeze-dried and sieved to a particle size below 250 μm before analysis. Freeze-drying is a gentle preservation method which was shown to have no significant influence on Hg speciation . In betw3) and hydrochloric acid (HCl)  were further purified by a two-stage sub-boiling distillation . Copper sulfate (CuSO4) pentahydrate (p.a.), glacial acetic acid (p.a.), methanol , and sodium hydroxide solution  were also purchased from Th. Geyer. Methylmercury chloride (99.8%), ethylmercury chloride , tetrahydrofuran , hydrofluoric acid , and sodium sulfate  were purchased from Sigma-Aldrich . The derivatization agent sodium tetra-n-propylborate , n-hexane, and dichloromethane (DCM)  were obtained from LGC Standards . Sodium thiosulfate pentahydrate (p.a.) was purchased from Merck .Ultrapure water (resistivity ≥18.2 MΩcm) was obtained from a Milli-Q Reference water purification system . Concentrated nitric acid (HNO201Hg-enriched mercury oxide (HgO) was purchased from Chemotrade  and 201Hg-enriched monomethylmercury solution (w(MMHg) = 5.1 mg/kg in 3:1 acetic acid/methanol mixture) from ISC Science . Dilutions of 6.9 μg/kg and 0.068 μg/kg 201Hg-enriched MMHg in methanol with dilute HCl (w ≈ 0.02 kg/kg) were prepared gravimetrically for spiking the samples. The 201Hg-enriched HgO was dissolved in dilute HNO3 and diluted further with ultrapure water to a HNO3 concentration of 1 mol/L. This stock solution was diluted with HNO3 (c = 1 mol/L) to a mass fraction of 20.7 mg/kg Hg, which was determined by reverse-ID ICP-MS prior to this study. A second dilution of 1.1 mg/kg in HNO3 (c = 1 mol/L) was prepared gravimetrically.201Hg-enriched spike and reference materials can be found in Table For validation of the analytical method, the sediment reference materials IAEA-456 , ERM-CC020 , and ERM-CC580  were used. Details on the 4 and HNO3 and DCM to directly extract MMHg into the organic phase for separation of inorganic Hg. The required volume of spike solution was added to the sample with the extraction solution and estimated for every sample to target an 201Hg/202Hg ratio of approximately 1. The actual amount of spike solution added was determined gravimetrically. A Na2S2O3 solution was used to back extract MMHg into the aqueous phase for propylation, and hexane was used for extraction of the propylated Hg species. A detailed description of the sample preparation procedure can be found in section The extraction procedure was based on a procedure described by Carrasco and Vassileva  using an201Hg-enriched MMHg spike. Three replicates were prepared for each sample and reference material per day, with exception of IAEA-456, which was prepared in three replicates on 1 day and only once on each of three other days due to the limited amount of this material. Each replicate was analyzed three times with GC/ICP-ToF-MS, with exception of the replicates of low concentrated samples , which were analyzed five times. For measurements with GC/ICP-Q-MS and GC/ICP-SF-MS, two replicates of ERM-CC580 and three procedural blanks with and one procedural blank without 201Hg-enriched MMHg spike were prepared. Each replicate of the unspiked blank and the reference material was analyzed three times, and each replicate of the spiked blanks five times.Very low concentrated sediment samples (IAEA-456 and samples 1 and 8) were prepared with a sample amount of 1 g; of all other samples, an amount of 0.25 g was used. Procedural blanks were prepared by following the whole sample preparation procedure without sediment and with (for determination of LOQ) and without 3 (w ≈ 0.01 kg/kg). This was necessary for two separate reasons. In ICP-MS, instrumental mass fractionation compromises the measured isotope ratio of the sample leading to a bias in results of ID analyses. This mass bias must be corrected for by analyzing a sample with a known isotope ratio . Due to the drift of plasma conditions, it is preferred to use an internal standard rather than a separate measurement. Because the MMHg spike is only enriched in 201Hg but not monoisotopic, Tl was used as an internal standard since it is similar in atomic mass and does not lead to spectral interferences with Hg isotopes. The wet aerosol was also used to introduce oxygen for complete combustion of the organic compounds eluting from the GC column. When working with dry aerosol, incomplete combustion of organic compounds  could lead to carbon deposits on the cones of the ICP-MS instrument, especially in splitless injection mode. The GC was equipped with a 30 m TG5-SilMS column  and a programmable temperature vaporizing injector (PTV) operated in splitless mode with an injection volume of 2 μL. The injection program used was a heating ramp of 10 °C/s from 250 to 400 °C. Further details on operating parameters can be found in Tables The analysis of MMHg was conducted using a Trace 1310 GC  coupled to an icpTOF 2R  via a heated transfer line  and a dual introduction injector. The straight inlet of the injector was connected to the transfer line, which consists of a sulfinert-coated stainless-steel tubing surrounded with a heating jacket. The GC column was fed into the stainless-steel tubing ensuring that the column end was located in the middle of the heating jacket. An additional gas line was also connected to the transfer line, so that the GC effluent was carried into the plasma within a gas flow of 0.6 L/min argon gas leading to rapid transfer and minimized band broadening. The additional gas was guided through a 3-m coiled stainless-steel tubing located inside the GC oven to allow for pre-heating of the gas. The second inlet of the injector was connected via tygon tubing to a 20-mL glass cyclonic spray chamber equipped with a glass concentric nebulizer introducing a constant flow of a 1 μg/L Tl standard solution in HNOFor coupling of the GC to an iCAP Q ICP-Q-MS and an Element 2 ICP-SF-MS , the same interface and GC parameters were used.w(Hg)MMHg) of the samples and reference materials was very large, between 0.125 and 75.5 μg/kg, resulting in very small peaks for low mass fractions and prominent peaks for high mass fractions. Therefore, the acquisition parameters could not be optimized for maximum isotope ratio precision, which is also largely dependent on the concentration/peak height of the analyte. This means that the resulting isotope ratio precision is rather a realistic one than a maximum precision attainable with the respective instrument for transient signals. Based on these considerations and tests with MMHg of natural isotope ratio, a compromising integration time of 100 ms per isotope was selected. All other instrumental parameters can be found in Tables The data acquisition parameters of the ICP-MS instruments, especially the integration (or dwell) time, were optimized to allow for a maximum signal-to-noise ratio (S/N) and a reasonable number of data points for low S/N. In general, an increasing integration time results in increasing S/N and decreasing number of data points per chromatographic peak. However, for isotope ratio precision, both a high S/N and a high number of data points per peak are favorable. It should be noted that the range of mass fractions of Hg being present as MMHg (201Hg/202Hg of the MMHg peak. The average baseline intensity and standard deviation (s) were determined from 50 data points each before and after the peak. The average baseline intensity was subtracted from every data point of the peak and peak data points were used for calculation when the intensity was larger than the average baseline intensity plus 3×s. The sum of these data points was used as the intensity of the respective isotope. A mass bias correction was performed following the Russell law using a Tl solution supplied by continuous liquid introduction as shown in Eqs. For consistent data treatment, the data from GC/ICP-MS analyses were evaluated using Microsoft Excel 365. With respect to the simultaneous detection, ICP-ToF-MS is similar to multicollector-ICP-MS (MC-ICP-MS), several approaches for calculating precise isotope ratios from transient MC-ICP-MS signals have been described, and linear regression (LRS) and peak area integration (PAI) methods have been found to be best suited , 35. The203Tl/205Tl isotope ratio  was determined by averaging the isotope ratio of every data point within the peak. The isotopic abundances used for calculating the true Tl isotope ratio  and isotopic masses of Tl  were taken from the Commission on Isotopic Abundances and Atomic Weights (CIAAW) [f) was then applied on the experimental 201Hg/202Hg isotope ratio :The experimental  (CIAAW) –39. The 201Hg/202Hg ratio of unspiked samples, and the mean corrected 201Hg/202Hg  was within the uncertainty range of the natural isotope ratio. The mass bias corrected Hg isotope ratios  of repeated measurements of one sample replicate were averaged and subsequently used for calculation of the mass fraction of Hg being present as MMHg (w(Hg)MMHg,sample) of the replicate as shown in Eq. DM).The applicability of this procedure was checked by analyzing the MMMHg) was calculated from the isotopic masses of Hg isotopes taken from CIAAW [MHg) was taken from CIAAW [w(MMHg)spike) was calculated based on the mass fraction of the stock solution given by the manufacturer and the gravimetric dilution. All masses including the mass of spike dilution added to the sample (mspike) and the mass of the sample (msample) were weighed on an analytical balance . Isotopic abundances of the sample  were taken from CIAAW [A201Hg,spike; A202Hg,spike) were taken from the manufacturer’s information.The molar mass of the spike MMHg MMHg was calculated for each replicate applying the Kragten method [sm) of the Hg isotope ratio and the dry matter fraction were used as the uncertainty components of the respective quantities. For plotting the results, all w(Hg)MMHg of the replicates of a sample were averaged, and a pooled uncertainty (upooled) was calculated from the uncertainties of the replicates  and the number of replicates (n) as shown in Eq. The measurement uncertainty of n method  for Eq. sm,replicates) to give the final combined uncertainty (ucombined) of w(Hg)MMHg as shown in Eq. The pooled uncertainty was then combined with the standard error of the mean of the replicates (U) with a coverage of approximately 95% was calculated by multiplying upooled with the coverage factor (k = 2). As procedure blank samples prepared in parallel to the sediment samples using the whole extraction and derivatization procedure did not show any MMHg peak and spiked procedure blank samples resulted in very low negative mass fractions, a blank correction could be omitted and no cross-contamination was observed.The expanded uncertainty , 2 mL conc. HCl (w ≈ 0.31 kg/kg), and 3 mL conc. HF (w ≈ 0.48 kg/kg) were added. The sample was digested in a CEM Discover SP-D microwave  at 200 °C for 20 min. The supernatant of the resulting digest was taken off and diluted to an average acid concentration of 1–2 mol/L. Hg was separated from the matrix using disposable PP anion exchange columns  filled with 1.5 mL AG1-X8 resin . The columns were washed with elution reagent (0.1 g/kg L-cysteine in 0.02 kg/kg HNO3) and 4×5 mL ultrapure water. Conditioning was done with 3 mL conc. HCl, 5 mL ultrapure water, and 5 mL HNO3 (c = 1 mol/L). Subsequently, the diluted sample digest was loaded onto the column, and the matrix was washed out with 3×5 mL HNO3 (c = 1 mol/L) and 5 mL ultrapure water. Hg was eluted with 2×5 mL L-cysteine (w = 0.1 g/kg) in HNO3 (w = 0.02 kg/kg). For analysis, the sample solutions were diluted with HNO3 (w = 0.02 kg/kg) to give an appropriate signal intensity. This matrix separation procedure was necessary to ensure matrix-matching between the samples and the ICP Hg standard solution used for mass bias correction because instrumental mass fractionation is strongly influenced by the sample matrix.For analysis of total Hg mass fractions, 0.5 g of sample was weighed into a 35-mL quartz microwave vessel fitted with a PFA liner, and the 201Hg/202Hg. The background Hg mass fraction was determined by analyzing three blank samples spiked with 201Hg-enriched Hg solution. The measurement uncertainty was calculated as described in the MMHg section with the background mass fraction as additional component.Total Hg analysis was conducted on an Element 2 sector-field ICP-MS  equipped with a self-aspirating glass concentric nebulizer  and a glass cyclonic spray chamber (20 mL). The instrumental parameters used can be found in Table srel of m = 3 repeated measurements of each of the n = 12 sample replicates) for ERM-CC580 representing samples with high w(Hg)MMHg and 4.7%  for IAEA-456 representing samples with low w(Hg)MMHg. The repeatability of the sample preparation was 1.8% (srel of n = 12 sample replicates) for ERM-CC580 and 4.6% (n = 6) for IAEA-456. The mean recoveries with their associated expanded uncertainties are (108 ± 7) % (n = 12) for ERM-CC580 and (105 ± 10) % (n = 6) for IAEA-456. The analytical method for the analysis of total Hg was validated by analyzing the reference materials ERM-CC580, ERM-CC020, and IAEA-456 in triplicate. The mean recoveries with their associated expanded uncertainties are (103 ± 3) % for ERM-CC580, (100 ± 3) % for ERM-CC020, and (107 ± 9) % for IAEA-456. The results of all analyses of the reference materials were not significantly different from the certified values as shown by En-values of < 1, determined according to DIN EN ISO/IEC 17043 [The analytical method for the analysis of MMHg was validated by analyzing the reference materials ERM-CC580 and IAEA-456 using the procedures for high and low concentrated samples, respectively. Recovery and repeatability were evaluated by performing analyses of the reference materials on four different days. The repeatability of the isotope ratio of one sample was 0.66% MMHg for a sample weight of 1 g) in case of MMHg and 0.01 mg/kg (expressed as w(Hg)total for a sample weight of 0.5 g) in case of total Hg. The mass fractions of all samples were above the respective LOQ.The limits of quantification (LOQ) of the complete analytical procedures were determined by spiking blank samples with the n = 12) determined with GC/ICP-ToF-MS, (109 ± 7) % (n = 2) with GC/ICP-Q-MS, and (108 ± 7) % (n = 2) with GC/ICP-SF-MS. The results clearly demonstrate the equivalence of the three investigated instruments, not only in terms of recovery but also regarding the uncertainty. However, w(Hg)MMHg is very high in ERM-CC580 resulting in high peaks and easy and precise isotope ratio calculation, regardless of the used instrument. An investigation of the uncertainty budget MMHg is largely dependent on the uncertainty of the mass fraction of the 201Hg-enriched MMHg spike solution and only to a small extent on the uncertainty of the experimental isotope ratio.The performance of GC/ICP-ToF-MS, GC/ICP-Q-MS, and GC/ICP-SF-MS for SSID of MMHg was compared based on the analysis of the ERM-CC580 reference material. The mean recoveries with their associated expanded uncertainties are (108 ± 7) % n = 1 determin201Hg/202Hg ratio depends on the S/N of the peaks, as shown in Fig. For a more thorough investigation in the region of lower concentrations, the hexane extracts of the reference material were diluted by a factor of 10 to 175 with hexane and analyzed again with all three instruments. For this, only one sample replicate was prepared. Quite surprisingly, the results were still similar, with only slightly wider uncertainty ranges for the highest dilution. The precision of the measured 201Hg/202Hg ratio (the only uncertainty component depending on the sample), the relative combined uncertainty of the resulting w(Hg)MMHg is 3%. It becomes clear that above a S/N of 10, the 201Hg/202Hg ratio precision is below 1.5%, which accounts for less than 25% of the combined uncertainty of w(Hg)MMHg or an increase of the combined uncertainty of 1% (to then 4%). Therefore, above S/N 10, the combined uncertainty is dominated by the uncertainty of the spike mass fraction, and small differences in isotope ratio precision are of no consequence. Below S/N 10, the influence of the measured 201Hg/202Hg ratio on the combined uncertainty increases, but this effect is in the same order of magnitude for all three instruments.Assuming an infinite precision of the measured w(Hg)MMHg. Although there is a spread of factor 5, the differences are most likely not significant in practice and are more probably due to random effects than to instrumental properties.The LOQ of the MMHg analysis for all three instruments was determined as described in the previous section. The resulting LOQ were 0.008 μg/kg, 0.016 μg/kg, and 0.003 μg/kg for ToF, Q, and SF instruments, respectively, calculated for a sample weight of 1 g and expressed as These results show that although reported for continuous liquid introduction, ICP-ToF-MS is not significantly superior to sequential Q- or SF-based instruments in terms of isotope ratio precision for SSID in transient signals, when one element is analyzed. The superiority of ICP-ToF-MS in measurements using conventional liquid introduction is apparently not readily transferable to transient signals. This is most probably due to the measurement principle of the ToF mass analyzer, which uses pulsed ion packages from the continuous ion stream generated by the ICP ion source . The dwew(Hg)MMHg between 0.180 and 39 μg/kg, and w(Hg)total of 0.056 to 126 mg/kg as shown in Fig. w(Hg) in samples 1 (km 57.4), 2 (km 62), and 8 (km 96) were below 0.5 μg/kg and 0.6 mg/kg for MMHg and total Hg, respectively, and thereby close to the postulated natural background, samples 3 (km 73.86) to 7 (km 88.91) show strongly elevated levels above 20 μg/kg and 9 mg/kg, respectively. This increase in between sampling sites 2 (km 62) and 3 (km 73.86) corresponds most probably to the former location of a chemical plant that produced mercury-based seed dressings and to the results of total Hg analysis in previous studies [w(Hg)MMHg is nearly constant over the course of Finow Canal with a drop after the confluence with the Oder-Havel Canal , but even approximately 16 km downstream of the confluence, a w(Hg)MMHg around one order of magnitude above natural background was found . For w(Hg)total, the maximum value of more than 100 mg/kg agrees with previous studies; however, back then the maximum was observed at site 3 (km 73.86), in close vicinity to the former chemical plant, with decreasing values in downstream direction. This could indicate a substantial downstream transport of contaminated sediment particles in recent years. In this context, it has to be noted that prior to a state horticultural show in 2002, sediment from the area downstream of the former chemical plant was removed by dredging possibly explaining lower w(Hg)total at site 3 (km 73.86). Also, for total Hg, a drop after the confluence of both canals can be seen.The SSID GC/ICP-ToF-MS and ID ICP-SF-MS analysis of the sediment samples from Finow Canal revealed  studies . Interesw(Hg) of MMHg and total Hg in samples 5 (km 77.94) and 8 (km 96) might be lower than at neighboring sites  because here the sampling could only be done in small bays that were characterized by very low streaming and considerably less amount of fine-grain sediment than observed at the other sampling sites. Therefore, suspended contaminated sediment transported with the streaming is less likely to be deposited there. However, for a detailed investigation, a representative sampling regarding area and depth would have to be conducted.The w(Hg)total and w(Hg)MMHg may vary considerably in the whole washing bed due to variations in the soil conditions. Because this is not the only washing bed where sediment from Finow Canal has been deposited in the past, it is to be expected that these washing beds contaminated with legacy Hg have been and will be contributing largely to atmospheric Hg0 emissions, as soils have been described as a source for Hg0 [The soil sample W, taken at the level of km 79.5, fits in with the overall trend of MMHg and total Hg contamination, with mass fractions of (41 ± 4) μg/kg and (117 ± 3) mg/kg, respectively. This is rather surprising because no further Hg/MMHg deposition could have taken place since Finow Canal sediment was placed there for the last time around 40 years ago. Apparently, the topsoil is still heavily contaminated, and no considerable changes have occurred. Of course, it has to be said that this statement is based on only one sample from one location, and  for Hg0 .0), MMHg, monoethyl-Hg (EtHg), and inorganic Hg (Hg2+), detected as Hg0, methylpropyl-Hg, ethylpropyl-Hg, and dipropyl-Hg, respectively. Several small peaks could be observed on the tailing of Hg2+, but they could not be identified and are probably artifacts produced during derivatization. It was shown in previous works that Hg0 is produced by reduction of analytes during sample preparation and/or analysis [0 present in the sample is lost during freeze-drying. Since it is described as rather unstable [The chromatogram of the soil sample Fig.  shows thanalysis , whereasunstable , EtHg mo201Hg-enriched MMHg. Comparing the 201Hg/202Hg of all species with the natural isotope ratio, it can be seen that all species but MMHg show an isotope ratio similar to the natural one. This indicates that no significant demethylation or reduction of MMHg takes place during sample preparation and analysis. Otherwise, the 201Hg/202Hg would have been shifted to values higher than natural.To investigate and exclude potential species transformation, the sample was spiked with w(Hg)total and w(Hg)MMHg (samples 1 and 2) and samples affected by industrial contamination.The amount of substance fraction of MMHg/total Hg of all samples was between 0.029 and 0.32% corresponding to the proposed natural fraction of 0.1–1% . No trenThe performance of ICP-ToF-MS for SSID GC/ICP-MS analysis of MMHg was compared with ICP-Q-MS and ICP-SF-MS. As opposed to the use in continuous liquid introduction, no improvement of isotope ratio precision was observed by using the ToF mass analyzer for transient signals when investigating only a limited number of isotopes. However, the (quasi-) simultaneous detection of the whole mass spectrum will probably lead to much better results of ICP-ToF-MS compared to the other single-collector ICP-MS instruments, when more than one isotope system is used. This could be investigated further, e.g., by additionally analyzing organotin and organolead species, which can also be derivatized using aqueous phase alkylation to be accessible for GC speciation , 14.w(Hg)MMHg determined in the samples show that natural (bio-)methylation of Hg is not hindered by the partially extremely high Hg concentration in the sediment. The analysis of one topsoil sample from a washing bed where contaminated sediment had been deposited around 40 years ago shows the same high values of both, w(Hg)total and w(Hg)MMHg. This means that Finow Canal sediments will act as a source for MMHg in this ecosystem for long time, whereas the sediment removed from the canal and deposited on washing beds contributes to atmospheric Hg0 emissions. It might be interesting to analyze also other environmental compartments  of this region to assess the overall status of MMHg distribution. Also, further investigation on the occurrence of EtHg could be interesting to see if it is produced within the sediment/soil or if it was released from the chemical plant because EtHg has been used as a component of seed dressings.Furthermore, the SSID GC/ICP-ToF-MS method was applied for the analysis of MMHg in sediments of Finow Canal, which is a heavily industrially polluted canal in Northern Germany. The high ESM 1(PDF 56 kb)


Background: Altered copper levels have been observed in several cancers, but studies on the relationship between serum copper and early-stage triple-negative breast cancer (TNBC) remain scare. We sought to establish a predictive model incorporating serum copper levels for individualized survival predictions.Methods: We retrospectively analyzed clinicopathological information and baseline peripheric blood samples of patients diagnosed with early-stage TNBC between September 2005 and October 2016 at Sun Yat-sen University Cancer Center. The optimal cut-off point of serum copper level was determined using maximally selected log-rank statistics. Kaplan-Meier curves were used to estimate survival probabilities. Independent prognostic indicators associated with survival were identified using multivariate Cox regression analysis, and subsequently, prognostic nomograms were established to predict individualized disease-free survival (DFS) and overall survival (OS). The nomograms were validated in a separate cohort of 86 patients from the original randomized clinical trial SYSUCC-001 (SYSUCC-001 cohort).Results: 350 patients were eligible in this study, including 264 in the training cohort and 86 in the SYSUCC-001 cohort. An optimal cut-off value of 21.3 μmol/L of serum copper was determined to maximally divide patients into low- and high-copper groups. After a median follow-up of 87.1 months, patients with high copper levels had significantly worse DFS (p = 0.002) and OS (p < 0.001) than those with low copper levels in the training cohort. Multivariate Cox regression analysis revealed that serum copper level was an independent factor for DFS and OS. Further, prognostic models based on serum copper were established for individualized predictions. These models showed excellent discrimination  and predictive calibration, and were validated in the SYSUCC-001 cohort.Conclusion: Serum copper level is a potential predictive biomarker for patients with early-stage TNBC. Predictive nomograms based on serum copper might be served as a practical tool for individualized prognostication. Triple-negative breast cancer (TNBC), characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2), is a heterogenic and aggressive subtype of breast cancer. TNBC is associated with a high risk of early relapse, a high degree of invasiveness, and poor prognosis . Early-sCopper is an essential element involving in many biological functions, including protein formation, enzyme activation, oxidation-reduction reactions, immunity development, and signaling pathways . Copper To address this need, we aimed to explore the relationship between baseline serum copper levels and the survival prognosis of patients with early-stage TNBC in this study, furtherly, we hoped to develop prognostic models combining serum copper levels and clinicopathological factor for individualized survival predictions and personalized decision-making.This study retrospectively analyzed the relationship between serum copper levels and the prognosis of patients with newly diagnosed TNBC at Sun Yat-sen University Cancer Center (SYSUCC) between September 2005 and October 2016. The study was approved by the ethics committee of SYSUCC (Registration number: B2021-218-01), written informed consent from patients was waived because of the retrospective nature of current study and the anonymous processing of patient information. We covered all personal data confidentially and conducted this study in accordance with the Declaration of Helsinki.in situ hybridization, 4) early-stage tumor, i.e., T1-4N0-3M0 according to the AJCC 2016 criteria (eighth edition) (Patients were eligible for inclusion in this study if they met the following criteria: 1) women with pathologically diagnosed breast cancer without distant metastasis  at the time of diagnosis, 2) age ≥18 years old, 3) ER/PR-negative, which was considered as <1% positive cells by immunohistochemistry (IHC) staining; HER2-negative tumor, which was defined as a score of 0 or 1 + on IHC or a score of 2 + on IHC without ERBB2 gene amplification on fluorescence edition) , and 5) Patients’ clinicopathological data were hand-retrieved from the electronic medical records system of SYSUCC. The peripheric blood samples collected at the time of diagnosis and before the initiation of any anti-cancer treatment were obtained from the tumor resource library of SYSUCC. The serum copper level of the participants was measured using the Copper Assay Kit (PAESA chromogenic method) of the Roche cobas 8,000 system .Patients were monitored every 3 months during the first 2 years, every 6 months during the subsequent 5 years, and once a year thereafter. The monitoring assessments included routine hematological and laboratory tests, menopausal status, breast and abdominal ultrasonography or computed tomography. X-ray and bone scans were performed annually.The primary endpoint was disease-free survival (DFS), which was defined as the time from the date of diagnosis to the date of first sign of disease progression or death due to any cause. The secondary endpoint was overall survival (OS), which was defined as the time from the date of diagnosis to the date of death due to any cause.maxstat” package of R software .Age was listed as mean with range, and categorical variables were represented as frequencies with percentages. The optimal cut-off value of serum copper level was determined by the maximally selected log-rank statistics using the “software . X-tile software . Survivaesiduals , and theAfter removing 75 patients with incomplete information, including 30 patients without Ki-67 index data, 38 patients without histological tumor grades, 4 patients without lymphovascular invasion data, and 3 patients without T-stage information , a total2 log-rank value, 11.5172). In the training cohort, the serum copper level was ≤21.3 μmol/L among 237 (89.8%) women and >21.3 μmol/L among 27 (10.2%) women. In the SYSUCC-001 cohort, 84 (97.7%) TNBC patients had a value ≤21.3 of the serum copper and 2 (2.3%) patients had the serum copper level >21.3 μmol/L.Using maximally selected log-rank statistics, we determined that a cut-off point of 21.3 μmol/L for the baseline serum copper level would maximally divide patients into two copper-stratified groups , i.e., tAfter a median follow-up of 87.1 months , we observed 64 DFS events and 51 deaths in training cohort, whose estimated 5-years DFS rates and 5-years OS rates were 79.6% (95% CI 74.8–84.6) and 83.7% (95% CI 79.3–88.4), respectively .p = 0.002) and OS (p < 0.001) than patients with low copper levels  in the univariate Cox regression analysis of OS in the training cohort  in the training cohort and 0.704 (95% CI 0.577–0.831) in the SYSUCC-001 cohort. Calibration curves for the DFS probability at 1-, 3-, and 5-years in the training and SYSUCC-001 cohorts both demonstrated satisfactory consistency between the nomogram-predicted and actual survival .The prognostic nomogram of OS also showed satisfactory discrimination, with an excellent C-index of 0.728 (95% CI 0.654–0.802) in the training cohort and 0.653 (95% CI 0.487–0.819) in the SYSUCC-001 cohort. The calibration curves for the probability of OS at 1-, 3-, and 5-years  also sugIn current study, we found that patients with early-stage TNBC were a heterogeneous population with different serum copper levels. According to maximally selected log-rank statistics, we determined that 21.3 μmol/L was the best cut-off value of serum copper to stratify patients into two different groups. Moreover, multivariate Cox analysis identified high serum copper level as an independent factor influencing survival. Prognostic nomograms incorporating serum copper level and other clinicopathological factors were developed, which showed excellent discrimination and good consistency between the nomogram-predicted survival and actual observations in both the training and SYSUCC-001 cohorts.As a catalytic cofactor and a structural component of various proteins and enzymes, copper is indispensable for organisms, and its homeostasis is crucial for many physiological functions . Copper In 1975, Schwartz firstly reported the potential value of copper in the diagnosis and prognostication of tumors . Gupte aHowever, some studies on breast and colon cancers have reported that the serum copper level was lower in tumor tissues than in normal tissues . Leila eBased on the stratification of serum copper levels, we established prognostic models that incorporated the serum copper level and other clinicopathological factors, including age and N stage, which is an indicator of metastasis . CompareThere are some limitations to our study. Firstly, as a retrospective study, selection bias and incomplete data were inevitable, an external validation rather than a same-institute validation might enhance the power of our predictive models. We minimized the risk of selection bias by enrolling all eligible patients to date and validating our results in a relatively separate cohort. Secondly, the serum cooper might be influenced by food, but we failed to obtain and explore information of patients’ dietary, so a prospective study to control these potential confounding factors and validate the prognostic value of our nomograms is warranted in future. Thirdly, we only explored the relationship between the baseline serum copper level at the time of diagnosis and early-stage TNBC. We did not measure the serum copper levels in healthy control subjects without TNBC, and monitor the dynamic changes in serum copper levels during the treatment of TNBC. Fourthly, this study was limited to patients from China, so the extension of our results to patients from other geographic regions or with other racial backgrounds should be done with caution. Finally, fundamental cytological studies and investigations of the underlying mechanisms are necessary to further explore the usefulness of serum copper levels in early-stage TNBC.In conclusion, we proposed an optimal cut-off value of baseline serum copper levels to maximally distinguish women with early-stage TNBC into different risk groups in current study. Based on this serum copper level cut-off, we developed and validated prognostic models for personalized survival predictions, resulting in satisfactory discrimination and good agreement between the nomogram-predicted and actual survival for survival in training and validations cohorts.